Enkephalin-δ opioid receptor signaling partly mediates suppression of LH release during early lactation in rats

Abstract

Gonadal function is often suppressed during lactation in mammals including rodents, ruminants, and primates. This suppression is thought to be mostly due to the inhibition of the tonic (pulsatile) release of gonadotropin-releasing hormone (GnRH) and consequent gonadotropin. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release, and kisspeptin mRNA (Kiss1) and/or kisspeptin expression in the ARC are strongly suppressed by the suckling stimuli in lactating rats. This study aimed to examine whether the central enkephalin-δ-opioid receptor (DOR) signaling mediates the suckling-induced suppression of luteinizing hormone (LH) release in lactating rats. Central administration of a selective DOR antagonist increased the mean plasma LH levels and baseline of LH pulses in ovariectomized lactating mother rats compared to vehicle-injected control dams on day 8 of lactation without affecting the number of Kiss1-expressing cells and the intensity of Kiss1 mRNA signals in the ARC. Furthermore, the suckling stimuli significantly increased the number of enkephalin mRNA (Penk)-expressing cells and the intensity of Penk mRNA signals in the ARC compared to non-lactating control rats. Collectively, these results suggest that central DOR signaling, at least in part, mediates the suppression of LH release induced by suckling stimuli in lactating rats via indirect and/or direct inhibition of ARC kisspeptin neurons.

Keywords: Arcuate nucleus; GnRH neuron; Kisspeptin neuron; Suckling stimulus.

Fig. 1.

Effects of central administration of naltrindole hydrochloride (NTI), a selective antagonist of δ-opioid receptors (DOR), on the suppression of luteinizing hormone (LH) pulses induced by suckling stimuli in ovariectomized (OVX) lactating rats during early lactation. Plasma LH profiles of individual OVX lactating rats, which were treated with the third ventricle (3V) injection of NTI (or vehicle) during early lactation (A). Blood samples were collected every 6 min for 3 h. Immediately after the first blood sampling, NTI or vehicle (timing indicated by arrows) was injected into the 3V. Arrowheads indicate the peaks of LH pulses identified by the PULSAR computer program. The mean plasma LH concentrations (B) and the baseline (C), frequency (D), and amplitude (E) of LH pulses in each group. Values are means ± SEM. The number in each column indicates the number of animals used. * Significant difference between NTI- and vehicle-treated rats (P < 0.05, Student’s t-test). N/A, not applicable.

Fig. 2.

Effects of central administration of NTI on the Kiss1 (kisspeptin gene) expression in the arcuate nucleus (ARC) of OVX lactating rats during early lactation. Kiss1-expressing cells in the ARC in representative OVX lactating rats treated with vehicle or NTI in the 3V 1 h before brain sampling (A). The insets indicate Kiss1-expressing cells (black arrowheads) at higher magnification. NTI administration did not affect the intensity of Kiss1 mRNA signals and the number of Kiss1-expressing cells in the ARC of OVX lactating rats (B). Scale bars = 100 µm. Values are means ± SEM. The number in each column indicates the number of animals used.

Fig. 3.

Effect of suckling stimuli on hypothalamic Penk (enkephalin gene) expression in OVX lactating rats during early lactation. Penk-expressing cells in the ARC (A), paraventricular nucleus (PVN) (B), and ventromedial hypothalamus (VMH) (C) in representative OVX lactating and non-lactating rats. The insets indicate Penk-expressing cells (black arrowheads) at higher magnification. The suckling stimuli increased the number of Penk-expressing cells and the intensity of Penk mRNA signals in the ARC (D), but not in the PVN (E) and VMH (F), of OVX lactating rats. Note that only Penk signal intensity is provided for the VMH because Penk-positive cells overlapped each other, thus, each Penk-positive cell was not distinguishable. Scale bars = 100 µm. Values are means ± SEM. The number in each column indicates the number of animals used. * Significant difference between lactating and non-lactating OVX rats (P < 0.05, Student’s t-test).